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Osteoclasts are multinucleated cells responsible for bone resorption. The differentiation of osteoclasts from bone marrow macrophages (BMMs) is induced by receptor activator of NF-κB ligand (RANKL). Osteoprotegerin (OPG), a decoy receptor of RANKL, inhibits osteoclastogenesis by blocking RANKL signaling. Here we investigated the degradation of OPG in vitro. Osteoclasts, but not BMMs, secreted OPG-degrading enzymes. Using mass spectrometry and RNA-sequencing analysis, we identified high-temperature requirement A serine peptidase 1 (HtrA1) as an OPG-degrading enzyme. HtrA1 did not degrade OPG pre-reduced by dithiothreitol, suggesting that HtrA1 recognizes the three-dimensional structure of OPG. HtrA1 initially cleaved the amide bond between leucine 90 and glutamine 91 of OPG, then degraded OPG into small fragments. Inhibitory activity of OPG on RANKL-induced osteoclastogenesis was suppressed by adding HtrA1 in RAW 264.7 cell cultures. These results suggest that osteoclasts potentially prepare a microenvironment suitable for osteoclastogenesis. HtrA1 may be a novel drug target for osteoporosis., Nagahiro Ochiai et al. report that osteoclasts, but not bone marrow macrophages, secrete a serine protease HtrA1 that can degrade osteoprotegerin in the bone microenvironment. Their results suggest HtrA1 recognizes the three-dimensional structure of osteoprotegerin and may function to prepare the microenvironment for osteoclastogenesis.

Objective Donepezil, an inhibitor of acetylcholinesterase (AChE) targeting the brain, is a common medication for Alzheimer's disease. Interestingly, a recent clinical study found that administration of this agent is associated with lower risk of hip fracture independently of falling, suggesting its direct effect on bone tissues as well. AChE has been reported to be involved in osteoblast function, but the role of AChE on osteoclastogenesis still remains unclear. We analyzed the effect of AChE and donepezil on osteoclastogenesis in vivo and in vitro. Methods Cell-based assays were conducted using osteoclasts generated in cultures of murine bone marrow macrophages (BMMs) with receptor activator of nuclear factor-kappa B ligand (RANKL). The effect of donepezil was also determined in vivo using a mouse model of RANKL-induced bone loss. Results Recombinant AChE in BMMs cultured with RANKL further promoted RANKL-induced tartrate-resistant acid phosphatase (TRAP)-positive osteoclast differentiation. RANKL also upregulated AChE expression in BMMs. RNA interference-mediated knockdown of AChE significantly inhibited RANKL-induced osteoclast differentiation and suppressed gene expression specific for osteoclasts. AChE upregulated expression of RANK, the receptor of RANKL, in BMMs. Donepezil decreased cathepsin K expression in BMMs and the resorptive function of osteoclasts on dentine slices. Donepezil decreased RANK expression in BMMs, resulting in the inhibition of osteoclast differentiation with downregulation of c-Fos and upregulation of Id2. Moreover, administration of donepezil prevented RANKL-induced bone loss in vivo, which was associated with the inhibition of bone resorption by osteoclasts. Conclusions AChE promotes osteoclast differentiation in vitro. Donepezil inhibits osteoclast function in vitro and prevents bone loss by suppressing bone resorption in vivo, suggesting the possibility that donepezil reduces fracture risk in patients with Alzheimer's disease.

Vitamin D3 is already found early in the evolution of life but essentially as inactive end products of the photochemical reaction of 7-dehydrocholestol with ultraviolet light B. A full vitamin D (refers to vitamin D2 and D3) endocrine system, characterized by a specific VDR (vitamin D receptor, member of the nuclear receptor family), specific vitamin D metabolizing CYP450 enzymes regulated by calciotropic hormones and a dedicated plasma transport-protein is only found in vertebrates. In the earliest vertebrates (lamprey), vitamin D metabolism and VDR may well have originated from a duplication of a common PRX/VDR ancestor gene as part of a xenobiotic detoxification pathway. The vitamin D endocrine system, however, subsequently became an important regulator of calcium supply for an extensive calcified skeleton. Vitamin D is essential for normal calcium and bone homeostasis as shown by rickets in vitamin D-deficient growing amphibians, reptiles, birds and mammals. From amphibians onward, bone is gradually more dynamic with regulated bone resorption, mainly by combined action of PTH and 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) on the generation and function of multinucleated osteoclasts. Therefore, bone functions as a large internal calcium reservoir, under the control of osteoclasts. Osteocytes also display a remarkable spectrum of activities, including mechanical sensing and regulating mineral homeostasis, but also have an important role in global nutritional and energy homeostasis. Mineralization from reptiles onward is under the control of well-regulated SIBLING proteins and associated enzymes, nearly all under the control of 1,25(OH)2D3. The vitamin D story thus started as inert molecule but gained an essential role for calcium and bone homeostasis in terrestrial animals to cope with the challenge of higher gravity and calcium-poor environment.

We have previously established a mouse coculture system of osteoblastic cells and spleen cells for examining osteoclast differentiation. In the present study, we examined the morphological features of the cell-cell contact between mouse spleen cells and osteoblastic cells in the co-cultures. Light microscopic investigations revealed that tartrate-resistant acid phosphatase (TRAP)-positive mononuclear and multinucleated spleen cells appeared in the vicinity of alkaline phosphatase (ALP)-positive osteoblastic cells. Ultrastructurally, spleen cells extended long cytoplasmic filopodia in all directions, by which spleen cells touched adjacent osteoblastic cells. The adjacent osteoblastic cells and spleen cells adhered to each other by forming electron-dense cytoplasmic materials on their inner leaflets of plasma membranes at cell-cell contact sites. Some adjacent osteoblastic and spleen cells made contact on the plasma membranes, forming an extracellular microenvironment. Coated pits were also formed on the plasma membranes facing this microenvironment. These morphological features of the cell-cell contact between osteoblastic cells and spleen cells indicate that there is internalization of organic components, i.e., receptor-mediated endocytosis in the contact sites between the two types of cells.

Osteoclast formation is regulated by balancing between the receptor activator of nuclear factor-κB ligand (RANKL) expressed in osteoblasts and extracellular negative regulatory cytokines such as interferon-γ (IFN-γ) and interferon-β (IFN-β), which can suppress excessive bone destruction. However, relatively little is known about intrinsic negative regulatory factors in RANKL-mediated osteoclast differentiation. Here, we show the paired-box homeodomain transcription factor Pax6 acts as a negative regulator of RANKL-mediated osteoclast differentiation. Electrophoretic mobility shift and reporter assays found that Pax6 binds endogenously to the proximal region of the tartrate acid phosphatase (TRAP) gene promoter and suppresses nuclear factor of activated T cells c1 (NFATc1)-induced TRAP gene expression. Introduction of Pax6 retrovirally into bone marrow macrophages attenuates RANKL-induced osteoclast formation. Moreover, we found that the Groucho family member co-repressor Grg6 contributes to Pax6-mediated suppression of the TRAP gene expression induced by NFATc1. These results suggest that Pax6 interferes with RANKL-mediated osteoclast differentiation together with Grg6. Our results demonstrate that the Pax6 pathway constitutes a new aspect of the negative regulatory circuit of RANKL-RANK signaling in osteoclastogenesis and that the augmentation of Pax6 might therefore represent a novel target to block pathological bone resorption.

The implantation of bone morphogenetic protein (BMP) into muscular tissues induces ectopic bone formation at the site of implantation. To investigate the mechanism underlying this process, we examined whether recombinant bone morphogenetic protein-2 (BMP-2) converts the differentiation pathway of the clonal myoblastic cell line, C2C12, into that of osteoblast lineage. Incubating the cells with 300 ng/ml of BMP- 2 for 6 d almost completely inhibited the formation of the multinucleated myotubes expressing troponin T and myosin heavy chain, and induced the appearance of numerous alkaline phosphatase (ALP)- positive cells. BMP-2 dose dependently induced ALP activity, parathyroid hormone (PTH)-dependent 3',5'-cAMP production, and osteocalcin production at concentrations above 100 ng/ml. The concentration of BMP-2 required to induce these osteoblastic phenotypes was the same as that required to almost completely inhibit myotube formation. Incubating primary muscle cells with 300 ng/ml of BMP-2 for 6 d also inhibited myotube formation, whereas induced ALP activity and osteocalcin production. Incubation with 300 ng/ml of BMP-2 suppressed the expression of mRNA for muscle creatine kinase within 6 h, whereas it induced mRNA expression for ALP, PTH/PTH-related protein (PTHrP) receptors, and osteocalcin within 24-48 h. BMP-2 completely inhibited the expression of myogenin mRNA by day 3. By day 3, BMP-2 also inhibited the expression of MyoD mRNA, but it was transiently stimulated 12 h after exposure to BMP-2. Expression of Id-1 mRNA was greatly stimulated by BMP-2. When C2C12 cells pretreated with BMP-2 for 6 d were transferred to a colony assay system in the absence of BMP-2, more than 84% of the colonies generated became troponin T-positive and ALP activity disappeared. TGF-beta 1 also inhibited myotube formation in C2C12 cells, and suppressed the expression of myogenin and MyoD mRNAs without inducing that of Id-1 mRNA. However, no osteoblastic phenotype was induced by TGF-beta 1 in C2C12 cells. TGF-beta 1 potentiated the inhibitory effect of BMP-2 on myotube formation, whereas TGF-beta 1 reduced ALP activity and osteocalcin production induced by BMP-2 in C2C12 cells. These results indicate that BMP-2 specifically converts the differentiation pathway of C2C12 myoblasts into that of osteoblast lineage cells, but that the conversion is not heritable.

IL-17 is a newly discovered T cell-derived cytokine whose role in osteoclast development has not been fully elucidated. Treatment of cocultures of mouse hemopoietic cells and primary osteoblasts with recombinant human IL-17 induced the formation of multinucleated cells, which satisfied major criteria of osteoclasts, including tartrate-resistant acid phosphatase activity, calcitonin receptors, and pit formation on dentine slices. Direct interaction between osteoclast progenitors and osteoblasts was required for IL-17-induced osteoclastogenesis, which was completely inhibited by adding indomethacin or NS398, a selective inhibitor of cyclooxgenase-2 (COX-2). Adding IL-17 increased prostaglandin E2 (PGE2) synthesis in cocultures of bone marrow cells and osteoblasts and in single cultures of osteoblasts, but not in single cultures of bone marrow cells. In addition, IL-17 dose-dependently induced expression of osteoclast differentiation factor (ODF) mRNA in osteoblasts. ODF is a membrane-associated protein that transduces an essential signal(s) to osteoclast progenitors for differentiation into osteoclasts. Osteoclastogenesis inhibitory factor (OCIF), a decoy receptor of ODF, completely inhibited IL-17-induced osteoclast differentiation in the cocultures. Levels of IL-17 in synovial fluids were significantly higher in rheumatoid arthritis (RA) patients than osteoarthritis (OA) patients. Anti-IL-17 antibody significantly inhibited osteoclast formation induced by culture media of RA synovial tissues. These findings suggest that IL-17 first acts on osteoblasts, which stimulates both COX-2-dependent PGE2 synthesis and ODF gene expression, which in turn induce differentiation of osteoclast progenitors into mature osteoclasts, and that IL-17 is a crucial cytokine for osteoclastic bone resorption in RA patients.

It is well established that osteoblasts and bone marrow stromal cells express osteoclast differentiation factor (ODF, also called RANKL) in response to several bone-resorbing factors to support osteoclast differentiation from their precursors. Osteoclast precursors which express RANK, a TNF receptor family member, recognize ODF/ RANKL through cell-to-cell interaction with osteoblasts/stromal cells, and differentiate into osteoclasts in the presence of M-CSF. Osteoclastogenesis inhibitory factor (OCIF, also called OPG) acts as a decoy receptor for ODF/RANKL. ODF/RANKL is responsible for inducing not only differentiation, but also survival and activation of osteoclasts. IL-1 and TNFα also play a major role in the pathogenesis of bone resorption induced by inflammation. IL-1 induced osteoclast differentiation by a classical ODF/RANKL-dependent mechanism, indicating that osteoblasts are essential for IL-1-induced osteoclast formation. In contrast, mouse TNFα strongly stimulated differentiation of M-CSF-dependent bone marrow macrophages (M-BMMφ) into osteoclasts without any help of osteoblasts/stromal cells. Osteoclast formation by TNFα was inhibited by antibodies against TNF receptor type 1 and 2 (TNFR1 and TNFR2), but not by OPG/OCIF, indicating that differentiation of M-BMMφ into osteoclasts by TNFα occurs by a mechanism independent of the ODF/RANKL-RANK interaction. IL-1 failed to induce differentiation of M-BMMφ into osteoclasts. More recently, we found that lipopolysaccharides (LPS)-induced bone loss did not occur in knockout mice of EP4, a subtype of PGE2 receptor. This indicates that EP4 signals are involved in the LPS-induced bone resorption. LPS appeared to induce osteoclast formation by two different pathways: one is an ODF/RANK-independent pathway involving TNFα. LPS induces TNFα production through toll-like receptor 4 (TLR4) in macrophages, which in turn directly acts on osteoclast progenitors through TNFR1 and TNFR2 to induce osteoclast differentiation. In this pathway, osteoblasts did not appear to be involved. The other pathway is the classical ODF/RANKL-dependent pathway. In the classical pathway, LPS induces PGE2 production through TLR4 in osteoblasts and macrophages, which in turn induces ODF/RANKL through EP4 in osteoblasts. ODF then binds ODF receptor (RANK) in osteoclast progenitors by cell-cell contact, which stimulates osteoclast differentiation. We conclude that osteoblasts/stromal cells are involved in not only physiological, but also pathological bone resorption via ODF/RANKL.

Thirty fertilized chick eggs preincubated for 0, 7 and 10 days on earth (10 eggs each) were flown in the space shuttle ‘Endeavour’ and further incubated for 7 days under microgravity. Twenty out of thirty eggs (910 ten-day-old; 10/10 seven-day-old; 110 zero-day-old) were recovered alive after landing. The only living embryo of the zero-day-old group died 24 days after launch, and was comparable to a 16-day-old embryo. The high mortality of the 0-day-old eggs appeared to be related to the specific inner structure of the egg. Simulation experiments performed on earth indicated that when yolk stayed in the albumen for more than 2 days, most of the embryos died. The subtle difference in specific gravity between the yolk (1.029) and albumen (1.040) plays a critical role in early chick embryogenesis.

Expression of p60c-src, the normal cellular counterpart of the transforming protein of Rous sarcoma virus (RSV), p60c-src, was examined in mouse and rat authentic osteoclasts and mouse osteoclast-like multinucleated cells (MNCs) formed in vitro. In co-cultures of mouse osteoblastic cells and spleen cells, the expression of p60c-src strikingly increased on day 5 in parallel with the appearance of MNCs in the presence of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). Immunohistochemical examination confirmed the high level expression of p60c-src in both mouse authentic osteoclasts and MNCs. Electron microscopic examination revealed that p60c-src was primarily localized on ruffled border membranes and vacuoles, but not on the clear zone in rat authentic osteoclasts. These results suggest that p60c-src is important in osteoclastic bone resorption.

Recombinant human interleukin 6 (IL-6), a lymphokine involved in the final differentiation of activated B-cells into antibody-forming cells, greatly suppressed proliferation and induced differentiation of murine myeloid leukemia cells (M1) into mature macrophage-like cells. When M1 cells were treated with IL-6, their growth was completely arrested as early as on day 2, and they were induced to differentiate morphologically into macrophage-like cells. Differentiation-associated properties such as phagocytic activity, adherence to the dish surface, Fc and C3 receptors, were also induced within 24 h by IL-6, and they reached their respective maximal levels on day 2 or 3. The potency of IL-6 in suppressing proliferation and inducing differentiation was much greater than that of 1α,25-dihydroxyvitamin D3 one of the most potent inducers of M1 cells. The present report indicates that IL-6 is involved in the differentiation of not only B-cells but also myeloid leukemia cells.

Plasma immunoreactive corticotropin-releasing factor (I-CRF) levels were determined by using a human CRF radioimmunoassay and an immunoaffinity procedure. The basal plasma I-CRF level in normal subjects was 6 +/- 0.5 pg/ml (mean +/- SD). We found that most plasma I-CRF levels were affected by stress, negative feedback, and circadian rhythm. Basal I-CRF levels were high in patients with Addison's disease, Nelson's syndrome, hypopituitarism stemming from pituitary macroadenoma, and CRF- and adrenocorticotropic hormone-producing tumors. A very low, but significant, amount of I-CRF was detected (1-3 pg/ml) in patients with Cushing's syndrome, in corticosteroid-treated patients, and in a patient with hypothalamic hypopituitarism. These results suggest that a major component of plasma I-CRF is of hypothalamic origin, however, other extrahypothalamic tissues cannot be ruled out as a minor source of plasma I-CRF.